The invention is directed to methods for the detection of target molecules in test solutions, particularly medically relevant molecules contained in bodily fluids. The methods described herein use specific nucleic acid ligands attached to solid supports at spatially discrete locations. The invention provides methods for detecting the binding of target molecules to nucleic acid ligands, and methods for using arrays of nucleic acid ligands in diagnostic medical applications.
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by Exponential Enrichment, termed the SELEX process, is described in U.S. patent application Ser. No. 07/536,428, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment,xe2x80x9d now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled xe2x80x9cNucleic Acid Ligands,xe2x80x9d now U.S. Pat. No. 5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled xe2x80x9cMethods for Identifying Nucleic Acid Ligands,xe2x80x9d now U.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX patent applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, abandoned in favor of U.S. patent application Ser. No. 08/430,709, filed Apr. 27, 1995, now U.S. Pat. No. 5,660,985, entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotides,xe2x80x9d that describes oligonucleotide containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, abandoned in favor of U.S. patent application Ser. No. 08/443,957, filed May 18, 1995, now U.S. Pat. No. 5,580,737, entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,xe2x80x9d describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, now abandoned, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2 Modified Nucleosides by Intramolecular Nucleophilic Displacement,xe2x80x9d describes oligonucleotide containing various 2xe2x80x2-modified pyrimidines.
Given the remarkable ability of nucleic acid ligands to be generated against many different target molecules, it would be desirable to have methods for using said ligands as a diagnostic tool. In particular, it would be desirable to attach a plurality of different nucleic acid ligands to a microfabricated solid support (a xe2x80x9cbiochipxe2x80x9d), and then assay the binding to said ligands of target molecules in a bodily fluid. The subject application provides such methods.
Methods are provided in the instant invention for obtaining diagnostic and prognostic nucleic acid ligands, attaching said ligands to a biochip, and detecting binding of target molecules in a bodily fluid to said biochip-bound nucleic acid ligands. In one embodiment of the instant invention, one or more nucleic acid ligands are chosen that bind to molecules known to be diagnostic or prognostic of a disease; these ligands are then attached to the biochip. Particular methods for attaching the nucleic acid ligands to the biochip are described below in the section entitled xe2x80x9cFabrication of the Nucleic Acid Biochip.xe2x80x9d The biochip may comprise either (i) nucleic acid ligands selected against a single target molecule; or more preferably, (ii) nucleic acid ligands selected against multiple target molecules. In the subject invention, the level of target molecule binding to nucleic acid ligands at defined spatial locations will be determined using bodily fluid from individuals known to have the disease for which that target molecule is known to be prognostic or diagnostic, and also using bodily fluid from healthy individuals. Bodily fluid from an individual seeking a prognostic report can then be assayed using the biochip, and comparison of the three sets of data will yield prognostic or diagnostic information for that individual.
In another embodiment, the specific nucleic acid ligands attached to the biochip bind specifically to all or a large number of components of blood plasma, or other bodily fluids, of a healthy individual. The pattern and level of binding of these ligands to their targets will then be determined by the methods disclosed below for healthy individuals, and also for individuals diagnosed with various medical conditions. A computer database of biochip binding data will then be established, with each disease giving rise to a unique xe2x80x9csignaturexe2x80x9d binding pattern. Bodily fluids from individuals desiring a prognostic or diagnostic report will then be contacted with the biochip, and the binding pattern obtained compared with the reference database.
In a related embodiment, the attached nucleic acid ligands will bind specifically to all or a large number of components of the blood plasma, or other bodily fluid, of an individual known to be suffering from a particular disease. Once the pattern and level of target molecule binding to this biochip has been determined for the individual suffering from the disease, this biochip can be used to screen individuals known to be at risk of developing this disease. This embodiment will be useful for diseases in which the target molecules are not found in the bodily fluid of healthy individuals, and the target molecules themselves are not yet identified (e.g., for viral, bacterial or parasitic infections where the causative agent has not yet been characterized at the molecular level).
In all the methods described in this invention, it is not necessary to know what each nucleic acid ligand is binding. The preceding two embodiments are particularly preferred, as they will allow for the early diagnosis of diseases for which there are no currently known assays, and for which diagnosis traditionally depends on the manifestation of overt disease symptoms. It will then be possible to identify the nucleic acid ligands that are binding target molecules of relevance, and thereby identify those target molecules. These embodiments will greatly expedite research into disease, and will provide many new target molecules that can be used in directed diagnostic and drug discovery programs. Furthermore, the nucleic acid ligands identified on the biochip that bind to these target molecules may themselves have potential for use as therapeutic agents.
In the most preferred embodiment, the biochip contains both types of nucleic acid ligands described in the previous two embodiments. Such a biochip will be able to detect or predict diseases where the diagnostic or prognostic criterion is (i) a change in the concentration of molecule(s) normally found in bodily fluid; and/or (ii) the presence of a molecule not normally found in a bodily fluid of a healthy individual, e.g., a viral protein. FIG. 1 shows the use of such a biochip. For the sake of clarity, only a simple 4xc3x974 array is illustrated; typical embodiments may use arrays that are 100xc3x97100 or greater.